J. Plant Physiol. Vol. 146. pp.

527-534 (1995)

Pigment and Structural Changes in Chlorella zOfingiensis

upon Light and Nitrogen Stress

ETAN BAR, MOSHE RISE, MARINA VISHKAUTSAN, and SHOSHANA (MALIS) ARAD
The Institutes for Applied Research, Ben-Gurian University of the Negev, P.O.B. 653, Beer-Sheva 84105, Israel

Received October 14, 1994 . Accepted January 19, 1995

Summary

The green alga Chlorella zofingiensis was found to respond very rapidly to exposure to combined condi-
tions of high light intensity and nitrogen deficiency by accumulation of secondary carotenoids. Accumu-
lation of secondary carotenoids was detected as early as 60 min after induction to light stress
(300Ilmol'm-2's-1) in a nitrogen-free medium. Canthaxanthin and astaxanthin (free and monoesters)
were detected 2 - 3 h later, and after an additional 12 h an astaxanthin diester also appeared. The accumu-
lation of total secondary carotenoids was linear in relation to time. After 24 h the main secondary carot-
enoids were the monoester of astaxanthin (about 50% of total secondary carotenoids) and canthaxanthin
(20 - 25 %). During the first 8 h of stress the content of the primary carotenoids iJ-carotene and lutein in-
creased but subsequently the content of both chlorophyll and the primary carotenoids was reduced. The
reduction in the content of the photosynthetic pigments was followed by a degradation of the thylakoids
and a reduction of the potential rate of photosynthesis (a), but not of Pmax • After 3 days under light stress
the chloroplast was modified to a chromoplast-like organelle, full of secondary carotenoids and free of
thylakoid membranes. Twelve hours after the induction of stress, lipid bodies containing secondary ca-
rotenoids appeared around the chloroplast and accumulated at the periphery of the cell. The profile of
the secondary carotenoids in the lipid bodies was similar to that in the chromoplast, both containing
double the amount of astaxanthin diester and half the amount of free astaxanthin as compared with total
cell carotenoids. After 3 days under stress, a hydrophobic layer rich in secondary carotenoids formed in-
side the cell wall. Our results suggested that the lipid layer functions as a light filter to reduce irradiation
of the cell components, to prevent photooxidative damage and to reduce water losses.

Key words: Astaxanthin, canthaxanthin, Chlorella, chromoplast, photosynthesis, green algae, keto carot-
enoids, lipid body, primary carotenoid, secondary carotenoid.

Abbreviations: TEM = transmission electron microscope; HPLC = high performance liquid chroma-
tography; a = initial slope of photosynthesis.

Introduction 1979; Ben-Amotz et aI., 1982; An et aI., 1989; Arad et al.,

Primary carotenoids constitute an integral part of the pho-
tosynthetic apparatus in higher plants and algae, and func-
tion as accessory pigments in the light-harvesting complex
and as protective agents against photooxidative damage (Sie-
fermann-Harms, 1987; Lichtenthaler, 1987; 1993). The ac-
cumulation of secondary carotenoids up to 10 % of the cell
dry weight is, however, a phenomenon unique to some spe-
a
cies of green algae, fungi and bacteria ohnson and Lewis,
1993; Avalos et aI., 1993; Morelli et aI., 1993). The synthe-
sis of secondary carotenoids in green algae is induced by
stress conditions, such as high-intensity irradiation and ni-
trogen deficiency (Donkin, 1976; Ben-Amotz et aI., 1982;
Czygan, 1982; Ben-Amotz and Avron, 1989; Arad et aI.,
1993; Rise et aI., 1994).
In the green microalgae Dunaliella bardawil and Tretepho-
lia aurea, under light stress in combination with nutrition
deficiency conditions that reduced the rate of cell division, iJ-

© 1995 by Gustav Fischer Verlag, Stuttgart

 528 ETAN BAR, MOSHE fuSE, MARINA VrSHKAUTSAN, and SHOSHANA (MALIS) ARAD
carotene, which usually appears as primary carotenoid, is ac-
cumulated and functions as a secondary carotenoid. No ke-
tocarotenoids were accumulated in those algae under these
stress conditions (Czygan and Kalb, 1966; Ben-Amotz et al.,
1982; Ben-Amotz and A vron, 1989). In contrast, in other
green microalgae, such as ChIarella emersonii, C. zojingiensis,
C. /usca, Coelastrum proboscideum, Scenedesmus and Haema·
tococcus sp., the most common secondary carotenoids pro-
duced are ketocarotenoids. The main ketocarotenoids found
in these species were canthaxanthin and astaxanthin (Good-
win and Jamikorn, 1954; Droop, 1955; Donkin, 1976). Asta-
xanthin appeared largely in ester form (Renstrom et al.,
1981; Yong and Lee, 1991; Grung et al., 1992; Rise et al.,
1994).
In Haematococcus and Dunaliella, secondary carotenoids
have been found in lipid bodies (Ben-Amotz et al., 1982;
Vechtel et al., 1992 a). In Haematococcus, the lipid bodies
seem to be arranged at the cell periphery and to form a lipid
film containing carotenoids, possibly acting as a light filter.
It has also been suggested that in some species of green mi-
croalgae the secondary carotenoids serve as precursors for
the synthesis of sporopollenin-like compounds in the algal
cell wall (Atkinson et al., 1972; Burczyk, 1987; Derenne et
al., 1992). This suggestion was based on four observations: i)
the appearance of sporopollenin upon stress, in parallel with
the synthesis of secondary carotenoids, ii) the accumulation
of secondary carotenoids at the cell periphery, iii) inhibition
of carotenogenesis that was accompanied by the formation
of sporopollenin-like compounds; and iv) in-vivo radiolabel-
i~g of secondary carotenoids that appear in the sporopolle-
mn.
The accumulation of secondary carotenoids by C. emerso-
nii and C. zojingiensis under a combination of high light in-
tensity and nitrogen deficiency has previously been observed
and characterized by Arad et al. (1993) and by Rise et al.
(1994). In C. zojingiensis, canthaxanthin, astaxanthin, an asta-
xanthin monoester and an astaxanthin diester were found
(Rise et al., 1994). Exposure of C. zojingiensis containing sec-
ondary carotenoids to high light intensity caused removal of
the ester groups from the astaxanthin; simultaneous chloro-
phyll degradation was not observed. De-esterification of the
astaxanthin esters was suggested to be a mechanism for light
protection against photo oxidation.
In order to further elucidate the functions of the secondary
carotenoids, we followed changes in the pigment profile and
structural modifications in C. zojingiensis subjected to high
light intensity and nitrogen deficiency, starting shortly after
induction of stress conditions.
Material and Methods
Alga and growth conditions
Chlorella zojingiensis (UTEX 32) was grown under constant fluo-
rescent light at an intensity of 50 I1mol· m-z's- I and at a controlled
temperature of 24 ± 2°C in conical tubes, 4cm in diameter, con-
taining 350mL of N-8 growth medium (Soeder et al., 1964) with
1 gr' L -I KN0 3, aerated with sterile air enriched with 0.2 % COz.
For induction of synthesis of secondary carotenoids the cells were
collected by centrifugation (750 X g for 10 min) and resuspended in
nitrogen free N-8 medium at an initial concentration of
2 '10 6 cells' mL -I for further growth under the same conditions but
under a light intensity of 300 I1mol· m-z·s- I .
Extraction and HPLC analysis ofpigments
Cells from 5 mL of culture containing 2-4.10 6 cells' mL -I were
collected by centrifugation (750 x g for 10 min), resuspended in
DMSO: ethanol (2: 3 v/v) and heated at 70°C in the dark for
20 min (Rise et a!., 1994). The pigment extract was filtered through
0045 11m filters and analyzed with a Varian 5000 LC HPLC system
with an L-4250 UVIVIS detector (Merck-Hitachi), a chromjet inte-
grator (Spectra Physics), and a 250 x 4 mm (id) Lichrospher 100 RP-
18,5 11m column (Merck) (Rise et a!., 1994). A stepwise elution pro-
gram with two solvents, methanol: water (75: 25 v/v) and ethyl ace-
tate, in a 30-min gradient at a flow rate of 0.6 mL' min -I was used
(Rise et a!., 1994). The principal absorbance detector was set at
470 nm, and peaks were identified according to retention times and
absorbance spectra (based on previous GC-MS and NMR identifica-
tion (Rise et a!., 1994).
A nalysis of the photosynthetic potential
Samples of cell suspension were diluted in duplicate to
2·106cell·mL- 1 in a growth medium containing ImM NaHC0 3
for analysis with an oxygen electrode (Rank Brothers, Bottisham,
UK). The oxygen electrode chamber was illuminated with a Hg
lamp at light intensities of 10-600 I1mol·s- l . m-Z.
Electron microscopy
Cells were collected by centrifugation (600 x g for 5 min) and re-
suspended in 3 % gluteraldehyde in 0.1 M phosphate buffer, pH 7.5,
for 3 h. The cells were then washed three times with phosphate buf-
fer, mixed into a 1.5 % agar solution at 40°C, and cooled rapidly in
ice. The cell-containing agar was cut into pieces, fixed in 1 % OS04
for 1 h, and washed three times with phosphate buffer. The samples
were dehydrated in ethanol followed by incubation in propylene
oxide. The samples were then embedded in Epon 812, sectioned
with an ultra-microtome to a thickness of 300 - 500 11m, and stained
with saturated uranyl acetate solution and then with 0.3 % lead cit-
rate. Specimens were examined with a Jeol 100CX transmission
electron microscope (TEM).·
Isolation oflipid bodies and chromoplasts
Cells from a 3-day-old culture, grown in a nitrogen-free medium
under a light intensity of 300 I1mol'm- z,s-t, were collected by cen-
trifugation (750 x g for 10 min) and suspended in 5 mL of a buffer
made up of OAM sucrose, 100mM HEPES-NaOH, 10mM KCI,
1 mM MgCI 2 , and 1 mM EDTA at pH 7.5 (Murphy and Cummins,
1989). The cells were frozen in liquid air and crushed with a pestle
and mortar in the presence of glass beads. Unbroken cells were re-
moved by centrifugation (750 x g for 20 min). The lipid bodies were
collected from the surface of the supernatant and washed twice with
the buffer. Chromoplasts were isolated according to Liedvogel et a!.
(1976) with the following modifications: the supernatant was cen-
trifuged at 16,500 x g for 30 min to separate the chloroplasts (found
in the pellet) from the chromoplasts (occurring as a band in the su-
pernatant). Since the culture was not synchronous, some of the cells
contained chloroplasts, which were removed with the pellet. The
band of chromoplasts was transferred into 50 mL of HEPES buffer
amended with 0.1 M sucrose and centrifuged again. This step was re-
peated twice. Pigments from the chromoplasts and the lipid bodies
were extracted in DMSO: ethanol (2: 3 v/v) and analyzed by
HPLC.
 Pigment and structural changes in Chlorella zojingiensls 529

:;l
A

"1
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o
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-S
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4

r
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I
20 30 .

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01. -<
I
"ii 016 .....;
I
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01.

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t-.
Fig. 1: HPLC chromatogram of pigments
extracted from C. zojingiensis grown under
~
-; "l
I
Gj 8
control conditions of low light intensity c:os .01 ~
<.J

(50 I1mol· m - 2. S-I) in nitrogen-containing

N-8 medium (A) and grown for 3 days un- -SQ
III
000 l I

der high light intensity (300 I1mol· m - 2 . S-I) ,.Q 0 .. ~

in nitrogen-free medium (B). 1 - lutein, < I !

2 - chlorophyll b, 3 - chlorophyll a, 4 - ()- "" 1 c

carotene, 5 - astaxanthin (free form), 6 - o~A

canthaxanthin, 7 - astaxanthin monoester I
(both peaks at retention times 28.32 and
28.98 min), 8 - astaxanthin diester.
. ,.:
RElEN110N nME (Min )
30

Statistics while no secondary carotenoids could be detected (Fig. 1).

In all experiments standard deviation (SD) was less than 5 % of
the mean. All results were statistically analysed for significance
(P < 0.05) by two-dimensional analysis of variance (Sokal and
Rohlf, 1981).
Results
Effect ofhigh light intensity and nitrogen deficiency on
pigment content during 24 h of stress
The algal culture was subjected to a high light intensity of
300 ~mol· m -2. S -I and nitrogen deficiency. At zero time
only photosynthetic pigments, including chlorophyll and
primary carotenoids ({3-carotene and lutein), were present
Chlorophyll content remained constant up to the 8th h of
stress, when it began to fall, reaching less than 30 % of its ini-
tial value after 24 h (Fig. 2). The concentrations of the pri-
mary carotenoids increased slowly, reaching a maximum af-
ter 8 -12 h and falling thereafter (Fig. 2). The ratio of {3-caro-
tene to lutein remained constant throughout the 24 h ({3-ca-
rotene/lutein 0.17 ± 0.013). Secondary carotenoids accumu-
lated over the 24 h period (Fig. 1, Fig. 2). After 4 h of stress,
three secondary carotenoids were detected: canthaxanthin
(17 % of total secondary carotenoids), free astaxanthin
(30 %), and astaxanthin monoester (53 %) (Table 1). By 12 h a
small amount of an astaxanthin diester was also detected,
and reached up to 12 % of the total secondary carotenoids af-
ter 24 h whereas the free astaxanthin decreased to less than
17 % (Table 1).
530 ETAN BAR, MOSHE RISE, MARINA VrSHKAUTSAN, and SHOSHANA (MALlS) ARAD

12 1.2
f; 6.0
i.e ;:;-
10 ..§ 1.0 .:..
5.0 i.e
'EOc ;;
;;" 0.8
..§
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..§ 8 Q
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8
'0 0.6
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6 "C
S '0
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"~ 2.0
..."e 4 ~
."
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"S 0.2 1.0
~
e
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2 "
<Il
;E
0.0 0.0
0 60 120 180 240 300
0
0 4 8 12 16 20 24 Time (min)

Time (h)
Fig. 2: Effect of high light intensity and nitrogen deficiency on
the content of primary (PC) and secondary (SC) carotenoids
and chlorophyll (Chi) in C. zojingiensis. A logarithmic culture
(2 '106 cells' mL -I) was transferred to nitrogen-free N-8 medium and
exposed to a light intensity of 300J.Lmol·m- 2 ·s- l • Samples were
taken after 0, 4, 8, 12 and 24 h under stress for pigment analysis.
Fig. 3: Effect of light stress and nitrogen deficiency on the content
of primary (PC) and secondary (SC) carotenoids during stress in C.
zojingiensis. A logarithmic culture (50 mL of 2 '10 6 cells' mL -I) was
transferred to nitrogen-free medium in a 500 mL beaker. The
beaker was placed in a water bath to prevent heating and the culture
was stirred using a magnetic stirrer. The culture was illuminated by
a light intensity of 800 J.Lmol· m -2. S -I and samples were taken for
pigment analysis at 0, 60, 90, 150, 210 and 270 min after exposure to
light stress.

Table 1: Pigment content in a secondary carotenoid-free C. zojin·

gtensis culture exposed to high light intensity and nitrogen defi- 120
ciency. The alga culture (4 . 106 cells' mL -I) was transferred to ni-
trogen-free N-8 medium and exposed to a light intensity of 300
J.Lmol . m-2 . S-I. Samples were taken after 0, 4, 8, 12 and 24 h of ex- 100
posure to the stress conditions for analysis of pigments (/-1g' mL -I).
Three experiments were done, each in duplicates with no signifi-
cant differences. ND - not detectable (less than 0.5 J.Lg' ml-I).
0;
C
. 80

Carotenoids Time (h) ...Q

<J

Q 60
0 4 8 12 24 t<

Canthaxanthin ND 0.04 0.2 0.43 1.95 40

Astaxanthin (free) ND 0.07 0.2
Astaxanthin monoester ND 0.13 0.31
Astaxanthin diesters ND ND ND
When the algae were illuminated at a higher intensity of
800 Ilmol· m -2. S-I secondary carotenoids could already be
detected by 60 min (Fig. 3). Accumulation of secondary ca-
rotenoids was linear with time, and after 4 h their content
was four to five times higher than that at 300 Ilmol· m- 2. S-I.
Primary carotenoids accumulated very rapidly during the
first hour, but thereafter their accumulation rate ceased. The
maximal amount of primary carotenoids was obtained after
150min under a light intensity of 800Ilmol'm-2's-1 vs.
8-12 hat 300 Ilmol· m- 2 's- l •
Effect ofhigh light intensity and nitrogen deficiency on
photosynthesis
The photosynthetic potential was measured in a culture of
C. zofingiensis growing under a light intensity of
300 Ilmol· m- 2 's- 1in a nitrogen-free medium for 24h (Fig. 4).
The initial slope a dropped before reduction in Pmax was
observed. After 24h a had decreased to 25-30% of its con-
0.57 1.57
0.90 4.78
0.06 1.14 20
0 4 8 12 16 20 24
Time (h)
Fig.4: Effect of light intensity of 300/-1mol'm- 2 's- 1 and nitrogen
deficiency on the photosynthetic potential in C. zojingiensis. A loga-
rithmic culture (2 '106 cells' mL -I) was transferred to nitrogen-free
N-8 medium and exposed to a light intensity of 300 J.Lmol·m- 2·s- l .
Samples in triplicates were taken after 0, 4, 8, 12 and 24 h under
stress for analysis of oxygen evolution under various light inten-
sities up to 600/-1mol·m- 2·s- l . Control Pm", was 3.4J.Lmole
02·s- l ·min - l .
trol level, while Pmax was reduced to 50% of the control
level.
In order to verify the protective role played by secondary
carotenoids in photosynthesis, the effect of a very high light
intensity (1500 Ilmol· m -2. S-I) on photosynthetic potential
in a pre-induced culture (containing secondary carotenoids)
was compared with that in a non-induced culture (Fig. 5).
The initial value of Pmax in the secondary carotenoid-contain-
ing culture was less than half of that in the secondary carote-

4.--------------------------------,
D
D D D analysis of variance.
30 60 90 120 150
Time (min)
Fig. 5: Effect of extremely high light intensity of (1500 !Lmol· m -2. S-I)
on Pmax in a culture of C. za/ingiensis containing secondary carotenoids
after 24h under high light intensity of (300 !Lmol·m- 2 ·s- 1) and nitro-
gen deficiency (0) and in a culture devoid of secondary carotenoids
(0).
noid-free culture. During the time of stress Pmax in the cul-
ture containing secondary carotenoids did not change,
whereas in the culture devoid of secondary carotenoids Pm• x
dropped within 20 min to about 15 % of its initial level, and
after 60 min no oxygen evolution could be detected.
Effect ofhigh light intensity and nitrogen deficiency on cell
structure
TEM was used to follow structural changes in the algal cell
during stress (high light intensity of 300 !lmol· m -2. S-1 and
nitrogen deficiency). The most significant changes were
found in the chloroplast: after 4 h exposure to stress, degra-
dation of thylakoids was observed and after 12 to 24 h the
grana structure of thylakoids had almost disappeared and the
content of thylakoid membranes was reduced (Fig. 6 a, b).
After 3 days (Fig. 6 c) the chloroplast had completely lost its
typical structure and took the form of an orange chromo-
plast-like organelle (as observed by light microscope) with-
out an inner membrane.
Two additional significant changes in the cell were ob-
served in response to stress. In specimens examined after 12
and 24 h, lipid bodies were seen to accumulate outside the
chloroplast close to the cell membrane (Fig. 6 a, b). In the
3-day specimen (Fig. 6 c), a lipid layer containing secondary
carotenoids was observed under the cell wall. A very thick
cell wall, which was found to be stable in concentrated sulfu-
ric acid (95 %), indicated the presence of an additional com-
ponent in the cell wall, such as sporopollenin-like molecules.
Pigment analysis of the lipid bodies and the chromoplasts
Lipid bodies and chromoplasts were isolated from a culture
grown for 3 days under a light intensity of 300 !lmol· m -2. S-1
in a nitrogen-free medium. Pigments were analyzed in the
lipid bodies, the chromoplasts and a cell extract (Table 2).
Chlorophyll was not found in the chromoplasts or the lipid
bodies, and only very small amounts of primary carotenoids
Pigment and structural changes in Chiarella za/ingiensis 531
Table 2: Profile of secondary carotenoids in total cell extract, lipid
bodies and chromoplasts. A secondary carotenoid-free culture of C.
za/ingiensis was exposed to high light and nitrogen deficiency and
analyzed after 3 days under stress. Concentration of each secondary
carotenoid was expressed as a percentage of the total secondary ca-
rotenoids. The experiment was repeated independently three
times, and single samples were analyzed each time. No significant
differences were found between the experiments as found by 2D
Carotenoids Lipid bodies Chromoplast Whole cell
Canthaxanthin 25.7 25.9 26.1
Astaxanthin (free) 4.9 6.1 10.4*
Astaxanthin monoester 49.8 47.2 52.2
Astaxanthin diestes 19.6 20.8 11.3"
* Significantly different (P < 0.05).
were detected. However, chlorophyll, /3-carotene, and lutein
were found in the total cell extract, probably from incom-
pletely transformed chloroplasts that were removed from
the chromoplast fraction (see Materials and Methods). No
significant differences were found between the profile of
secondary carotenoids of the lipid bodies and that of the
chromoplasts (Table 2): in both, the astaxanthin diester
constituted about 20 % of the total amount of secondary
carotenoids (vs. about 11 % in the total cell extract). The
relative amount of free astaxanthin in the total secondary
carotenoids in the lipid bodies and in the chromoplasts was
about half in the total cell extract. No significant differences
were found between the relative amounts of total astaxanthin
and canthaxanthin in the lipid bodies, chromoplasts and to-
tal cell extract. In all fractions astaxanthin amounted to 74.1
± 2 % of total secondary carotenoids and canthaxanthin to
25.9 ± 2 %. In an older culture (~ 7 days) no differences
were found in the profile of secondary carotenoids among
the total cell extract, lipid bodies and chromoplasts, and no
chlorophyll or primary carotenoids were detected (results
not shown).
Discussion
Carotenoids playa highly effective role in protecting pho-
tosynthetic pigments, enzymes, membranes and nucleic
acids against photooxidative damage (Lichtenthaler, 1987;
Siefermann-Harms, 1987). During photosynthesis, oxygen
radicals are formed even under relatively low light inten-
sities. Under high irradiation the photosynthetic apparatus
does not sufficiently utilize light energy, and the excess
energy leads to the formation of highly active oxygen mole-
cules. In this case the primary carotenoids cannot scavenge
the radicals sufficiently, and additional mechanisms there-
fore are required for eliminating radicals or for reducing the
illumination reaching the cell components under such condi-
tions. In green microalgae such as Chlorella and Haematococ-
cus, secondary carotenoids that accumulate in response to
stress conditions may serve as agents protective against the
effects of photooxidation (Rise et al., 1994; Vechtel et al.,
1992 b).
532 ETAN BAR, MOSHE RISE, MARINA VISHKAUTSAN, and SHOSHANA (MALIS) AKAD

In our study, secondary carotenoids that were not present
in algae grown under regular conditions (Fig. 1) appeared
very shortly after exposure to combined stress conditions of
high light intensity and nitrogen deficiency (Fig. 3), which is
evidence for a rapid mechanism for protecting the photosyn-
thetic apparatus against irradiation damage. Significant sup-
port for the premise that secondary carotenoids provide pro-
tection against light stress comes from the fact that in cells
containing secondary carotenoids and exposed to high irra-
diation, equivalent to the intensity of sunlight, a sharply re-

~ .~ .
." ~ .. ""
• V' ,J
'

'- ~:'J ' ': 6 ~

12
6a
Fig. 6: Transmission electron micrographs of C. zojingiensis subjected to a high light intensity of 300 limol· m -2. S-1 and nitrogen defi-
ciency. 6 a - Cell structure in speciemens subjected to stress for 0, 4, 12 and 24 h, 6 b - Sample taken after 24 h of stress at a higher magnifi-
cation, 6 c - Sample taken after 72 h of stress. S-starch granules, L - lipid bodies, m - mitochondria, g - grana, arrows - thylakoids. Bars
are 2 lim.

duced photosynthetic potential in cells lacking secondary ca-
rotenoids but not in cells in which secondary carotenoids
had accumulated was found (Fig. 5). Indeed, in parallel to the
accumulation of secondary carotenoids, the chloroplast lost
its function due to the degradation of the thylakoids and the
\ photosynthetic apparatus against photooxidation. During
:~,
:.,..~
6c
Pigment and structural changes in Chlorella zoJingiensis 533
reduction in photosynthetic pigments. Since photosynthesis
is almost inactive during the period that secondary carot-
enoids continue to accumulate, it seems that they protect
photosynthesis only at the early stages of the stress. The ac-
cumulation of high concentrations of secondary carotenoids
during the later stages of stress suggests that they play an ad-
ditional role in helping the cell to survive during a long pe-
riod of stress by preventing irradiation damage to cell com-
ponents and not only protecting photosynthesis against pho-
tooxidation but also preventing irradiance damage to other
cell components.
We suggest that the response to stress is made up of three
stages. During the first stage (0 - 8 h, depending on light in-
tensity) both primary and secondary carotenoids protect the
the second stage (4 -12 h), when the content of primary ca-
rotenoids is reduced and secondary carotenoids begin to ac-
cumulate, the secondary carotenoids replace the primary ca-
rotenoids in protecting photosynthesis. During the third
stage (12 h and more) a lipid layer containing secondary ca-
rotenoids surrounds the cell to form a hydrophobic light fil-
ter, as previously suggested by Santos and Mesquita (1984)
and by Hagen et al. (1994). The secondary carotenoids may
also function as a substrate for the synthesis of sporopollenin
in the algal cell wall, as suggested by Derenne et al. (1992),
Atkinson et al. (1972) and Burczyk (1987). The thick and
stable cell wall found in the 3-day-old culture (Fig. 6 c) pos-
sibly contains a sporopollenin-like substance.
The transportation of the secondary carotenoids from the
chloroplast to the cell periphery is possibly accomplished by
lipid bodies. Formation of a lipid layer enriched with carot-
enoids was observed clearly in the 24-h and 72-h specimens
(Fig. 6). This lipid layer seems to function as a sunshade filter
to reduce irradiation of the cell components and to prevent
formation of oxygen radicals. It may also function as an ef-
fective hydrophobic layer that reduces water losses upon de-
hydration or salinization of the water body (occurring upon
evaporation).
Acknowledgements
The authors wish to thank Dr. S. Guttman from the Biologial
Services, Electron Microscopy Department, at the Wiezmann In-
stitute of Sciences, Israel for technical assistance in the microscopy
study and to Ms I. Mureinik and Ms D. Imbar for editing the manu-
script.
The work was partially supported by the Ben-Gurion Foundation
of the Ministry of Science and Arts of Israel.
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